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Use of embryonated chicken egg as a model to study the susceptibility of avian influenza H9N2 viruses to oseltamivir carboxylate
Journal of Virological Methods 224 (2015) 67–72
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Use of embryonated chicken egg as a model to study the susceptibility of avian influenza H9N2 viruses to oseltamivir carboxylate Deeksha S. Tare, Shailesh D. Pawar ∗ National Institute of Virology-Microbial Containment Complex, 130/1, Sus Road, Pashan, Pune 411021, India
a b s t r a c t Article history: Received 11 January 2015 Received in revised form 13 August 2015 Accepted 13 August 2015 Available online 20 August 2015 Keywords: Antivirals Oseltamivir Avian influenza H9N2 Embryonated chicken eggs
Avian influenza (AI) H9N2 viruses are endemic in many bird species, and human infections of H9N2 viruses have been reported. Oseltamivir phosphate (Tamiflu® ) is the available antiviral drug for the treatment and prophylaxis of influenza. There are no reports of use of embryonated chicken egg as a model to study susceptibility of AI viruses to oseltamivir carboxylate (OC), the active metabolite. The present study was undertaken to explore the use of embryonated chicken eggs as a model for testing OC against the AI H9N2 viruses. A total of three AI H9N2 viruses, isolated in poultry in India, were used. Various virus dilutions were tested against 14 g/ml of OC. Three methods, namely (1) the in vitro virus–drug treatment, (2) drug delivery and virus challenge by allantoic route, and (3) drug delivery by albumen route and virus challenge by allantoic route were explored. The viruses were also tested using the fluorescence-based neuraminidase inhibitor (NAI) assay. There was significant inhibition (p < 0.05) of the H9N2 viruses in presence of OC. The infectious virus titers as well as hemagglutination titers were significantly lower in presence of OC as compared to controls. The in vitro treatment of virus and drug; and drug and virus delivery at the same time by allantoic route showed significantly higher inhibition (p < 0.05) of virus growth than that by the albumen route. In the NAI assay, the half maximal inhibitory concentration (IC50 ) values of the H9N2 viruses were within the standard range for known susceptible reference virus. In conclusion, the H9N2 viruses used in the study were susceptible to OC. Embryonated chicken egg could be used as a model to study susceptibility of AI viruses to antiviral drugs. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Influenza viruses are single stranded, negative-sense RNA viruses, which belong to the family Orthomyxoviridae. The world experienced the most recent influenza pandemic in the year 2009 caused by the H1N1 virus. Avian influenza (AI) viruses such as H5N1, H7N9, and H9N2 are emerging and are potential threats to human and animal health. In addition to this, there are reports of resistance of influenza viruses to the antiviral drugs such as oseltamivir (Monto et al., 2006). Therefore, it is necessary to screen existing as well as emerging influenza viruses against the available antiviral agents. Oseltamivir (oseltamivir phosphate) (Tamiflu® ) is an orally administered antiviral drug, recommended by the World Health Organization (WHO) for use in the clinical management of pandemic and seasonal influenza virus infections of varying severity.
∗ Corresponding author. E-mail address: [email protected] (S.D. Pawar). http://dx.doi.org/10.1016/j.jviromet.2015.08.009 0166-0934/© 2015 Elsevier B.V. All rights reserved.
Oseltamivir carboxylate (OC) is the active metabolite of oseltamivir, and is a transition-state analog of sialic acid that is a potent selective inhibitor of influenza A and B virus neuraminidases (Hayden, 2005). There have been increasing speculations about the pandemic potential of AI H9N2 viruses (Paul, 2008). There are reports of prevalence of H9N2 viruses in avian species from several parts of the world, and also from poultry in India (Pawar et al., 2012a; Nagarajan et al., 2009). Human infections of H9N2 virus have been reported from China, Hong Kong, and Egypt (Peiris et al., 1999; Butt et al., 2005; World Health Organization, 2015), and seroprevalence of antibodies against H9N2 viruses has also been reported (Xiong et al., 2014; Zhou et al., 2014; Huang et al., 2013; Pawar et al., 2012b; Hadipour, 2011; Guo et al., 1999). The existing methods for studying the susceptibility of influenza viruses to oseltamivir include use of neuraminidase inhibition assay and analysis of neuraminidase (NA) gene sequence for the markers of drug resistance to the drug (Hurt et al., 2004; McKimm-Breschkin, 2000). The use of tissue culture, mice, and ferrets for antiviral studies on influenza viruses has been reported
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(Govorkova et al., 2001; Govorkova et al., 2007). Moreover, no correlation between the susceptibility of H5N1 viruses by NAI assay and the protection offered by oseltamivir in mice (in vivo) was found (Govorkova et al., 2009). Embryonated chicken eggs have been conventionally used for isolation and propagation of influenza viruses (World Health Organization, 2002). The antiviral activities of amantidine, rimantidine, and zanamivir against influenza viruses have been studied using the egg model (Haertl et al., 2004; Sauerbrei et al., 2006). In the reported studies, the drug delivery was via the albumen route and virus challenge was by the chorioallantoic membrane. The use of the egg model by exploring the allantoic route for virus inoculation and the albumen route for drug delivery has been reported (Wang et al., 2008). However, there are no studies comparing various routes for drug delivery of OC in eggs for AI viruses. The present work was undertaken to study the susceptibility of three isolates of AI H9N2 viruses from India to OC, and to explore three methods for virus and drug treatment and delivery using embryonated chicken egg as a model. 2. Materials and methods 2.1. Viruses used AI H9N2 viruses isolated from India, namely, A/chicken/India/ WB-NIV1057183/2010 (H9N2) [H9N2-WB-1057183] (GenBank: JX310069), A/chicken/India/WB-NIV1057209/2010 (H9N2) [H9N2-WB-1057209], and A/chicken/India/JH-NIV 124248/2012 (H9N2) [H9N2-JH-124248], were used in the study. 2.2. Virus propagation and detection The virus stock was prepared in embryonated chicken eggs by inoculating the virus by the allantoic route. The eggs used in all the experiments were 10-days-old at the time of inoculation and 13-days-old at the time of completion of experiment. The eggs were incubated for 72 h at 37 ◦ C, and were observed daily. After completion of the incubation, the embryos were chilled overnight at 4 ◦ C. The allantoic fluid was harvested, and hemagglutination (HA) assay was performed using 0.5% turkey red blood cells (World Health Organization, 2002). The virus stock was stored at −80 ◦ C till further use. The clinical end point was detection of virus by HA assay as a measure of virus growth in eggs. The study was conducted in accordance with the institutional guidelines. 2.3. Preparation of antiviral drug stock solution and toxicity testing The antiviral drug OC was kindly provided by Hoffmann-La Roche, Basel, Switzerland. A suspension of the drug was made in phosphate buffered saline (PBS) (pH 7.2). Peak plasma concentration of OC after a 75 mg dose in humans has been reported to be 0.35 g/ml (Hayden, 2005). Toxicity of OC was tested in eggs by inoculating 14 g/ml, 28 g/ml, 56 g/ml, and 112 g/ml of OC. The inoculated eggs were observed for 72 h. The embryos were decapitated, and fixed in formalin for a minimum period of 48 hs for histopathological analysis. Whole sections of the embryos were processed for paraffin embedding, sectioned at 4 m and stained with hematoxylin and eosin; and were examined for histopathological changes. 2.4. 50% egg infectious dose in presence and absence of OC The 50% egg infectious dose (EID50 ) titer of H9N2 virus was determined in the presence and absence of 14 g/ml of OC. The
Fig. 1. Schematic diagram of 10-days-old embryonated chicken egg showing the allantoic and albumen routes used for virus and drug inoculation.
virus was serially diluted tenfold (undiluted to 10−9 ) and treated by the in vitro drug treatment method (Song et al., 2007; Rajik et al., 2009). Each dilution was inoculated in ten eggs. Eggs showing HA titer ≥ 2 HA units were considered positive, while those showing no titer were negative. The EID50 was calculated using the Reed and Muench method (Reed and Muench, 1938). The mean log HA titers for individual dilutions were also compared. The experiment was performed three times. 100 EID50 virus was then used for the further experiments using three different methods.
2.5. In ovo antiviral assays Various drug concentrations of OC, 1.75 g/ml, 3.5 g/ml, 7 g/ml, 14 g/ml, 28 g/ml, 56 g/ml, and 112 g/ml were tested against 100 EID50 virus, using the in vitro drug treatment method (described below), to determine the concentration of drug required for complete inhibition of virus growth. Each drug concentration was inoculated in ten eggs. Drug concentrations 1.75 g/ml, 3.5 g/ml, and 7 g/ml did not show any significant drop in the virus HA titers after treatment as compared to the untreated controls (p > 0.05). OC concentrations of 14 g/ml and above showed complete inhibition of the virus. Therefore, 14 g/ml of OC was used for the in ovo antiviral assays. The following three methods were then used for the in ovo antiviral assays: (a) In vitro treatment of virus and drug: Equal volumes of the drug and virus were mixed and incubated for 1 h at 37 ◦ C. This mixture (0.2 ml virus + 0.2 ml drug) was inoculated into the allantoic cavity. (b) Virus and drug delivery by the allantoic route: In the allantoic cavity, 0.2 ml virus suspension was inoculated. In addition, 0.2 ml drug was administered at two time points [namely 0 h (ALL 0 h) and 2 h (ALL 2 h)] by the allantoic route. (c) Drug delivery by the albumen route: In the allantoic cavity, 0.2 ml virus was inoculated and 0.2 ml drug was administered, through albumen at two time points; 0 h (ALB 0 h) and 2 h (ALB 2 h) (Fig. 1) (Wang et al., 2008). Each treatment group, including the virus control group, consisted of ten eggs. There was no drug administration in the virus controls, PBS was used instead of OC. Total controls, which had no virus or drug inoculated, were also included in each experiment. The inoculated eggs were incubated at 37 ◦ C for 72 h. The eggs were observed after an interval of 24 h till 72 h. At the end of 72 h, the eggs were chilled at 4 ◦ C overnight. The allantoic fluids were harvested, and tested by the HA assay.
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Table 1 50% egg infectious dose (EID50 ) titers of drug-treated and untreated H9N2 viruses. AI H9N2 virus strains
H9N2-WB-1057183
Egg infectious dose 50% (EID50 ) titersa Untreatedb
Treatedc
107.57
103.40 103.47
107.61 109.39 H9N2-WB-1057209
H9N2-JH-124248
1010.00 1010.00
103.30 104.35 106.36
109.45 108.42
104.31 103.70
109.84 109.49
104.54 106.10
a 50% infectious titers calculated by the Reed and Muench method. Virus dilutions from undiluted to 10−10 . Each experiment was performed three times. b Untreated controls. No oseltamivir carboxylate was used. c Treated with 14 g/ml oseltamivir carboxylate. The treated groups showing significant difference as compared to the untreated ones (p < 0.05) depicted by boldface font (Independent sample t-test)
2.6. Statistical analysis
Fig. 2. Inhibition of virus growth by oseltamivir carboxylate using three treatment methods. The mean of log HA titers of the allantoic fluids harvested from the treated and untreated groups plotted against the various treatment methods used. In vitro drug–virus treatment method, and inoculation of the drug and virus at the same time by allantoic route (ALL 0 h) showed significant inhibition (p < 0.05) in the virus titers.
Independent sample t-test for the comparison of virus growth inhibition between treated and untreated groups was performed in the software “SPSS” (IBM). The log mean HA titers from inoculated eggs were calculated in “Microsoft Excel” and the difference in the HA titers was determined by t-test in the “EpiInfo” software (Centers for Disease Control, USA).
virus by the in vitro treatment method. The other methods of treatment, namely the ALL 2 h, ALB 0 h, and ALB 2 h showed inhibition as compared to the untreated controls, but it was not significant when compared to the in vitro and ALL 0 h treatment methods (Fig. 2).
2.7. Fluorescent neuraminidase inhibition (NAI) assay The inhibitory effect of OC on the viruses was also tested by the fluorescence-based NAI assay as per the method described by Hurt et al, 2004 to screen susceptibility or resistance. The viruses A/Fukui/20/2004 wild type H3N2 and A/Fukui/45/2004 (H3N2) 119 V mutant (kindly provided by Dr. Aeron Hurt at the WHO Collaborating Center for Reference and Research on Influenza, Melbourne, Australia) were used as reference standards for susceptibility and resistance, respectively. The plots and the IC50 values were calculated for the samples and standards by using the curve fitting software JASPR (CDC, USA). 3. Results
3.4. Fluorescent NAI assay The IC50 values of all the three H9N2 viruses were 0.72 nM, 3.66 nM, and 0.26 nM for H9N2-WB-1057183, H9N2-WB-1057209, and H9N2-JH-124248 viruses, respectively, which were in the range (0.01 nM–5.0 nM) of the OC sensitive standard strain A/Fukui/20/2004 (H3N2). This indicated that all three tested H9N2 viruses were sensitive to OC. H9N2-WB-1057209 virus showed the higher IC50 value among the three viruses. The reference resistant strain A/Fukui/45/2004 (H3N2) had IC50 values in the range 23 nM–378 nM (Fig. 3) (International Society for Influenza and other Respiratory Virus Diseases (ISIRV) Antiviral group (AVG)).
3.1. Toxicity testing
4. Discussion
There was no toxicity of OC in the eggs, as indicated by the absence of mortality and abnormalities in the histopathology of the embryos.
Oseltamivir phosphate has been shown to be effective in the experimental infection of mice by H9N2 virus, and also by using a modified enzyme-linked immunoassay and NAI assays (Leneva et al., 2000; Govorkova et al., 2001). However, there are no studies exploring various routes of virus and drug delivery in eggs for testing OC against AI H9N2 viruses. The previous studies in eggs used human viruses, and the drug oseltamivir phosphate and not the active OC (Heartl et al., 2004; Sauerbrei et al., 2006; Wang et al., 2008). However, in the present study, OC, which is the active metabolite form, has been used. In ovo experiments are at the borderline between in vitro and in vivo studies, and hence do not conflict with ethical and legal aspects of animal protection (Sauerbrei et al., 2006; Wang et al., 2008) When infectious virus titers (EID50 ) of three viruses with and without OC were compared (Table 2), H9N2-WB-1057209 virus dilutions from 10−1 to 10−3 did not show significant inhibition in the mean log titers. This might be due to the fact that this virus showed higher IC50 value i.e., 3.66 nM, as compared to the other two
3.2. 50% egg infectious dose in presence and absence of OC When the various dilutions of the three viruses were subjected to treatment (by the in vitro drug virus treatment method), there was significant reduction (p < 0.05) in the infectious virus titers (EID50 ) and also in the mean log HA titers in the presence of OC, as compared to controls (Table 1, 2). 3.3. In ovo antiviral assays In the further experiments for exploring different routes, the in vitro drug–virus treatment method, and inoculation of the drug and virus at the same time by allantoic route (ALL 0hr) showed significant inhibition (p < 0.05) of the virus, as compared to the other drug treatment methods. There was a complete inhibition of the
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Fig. 3. Half maximal inhibitory concentration (IC50 ) of H9N2 viruses in neuraminidase inhibition assay. Output graphs obtained for the neuraminidase inhibition assay, from the software JASPR. (a), (b), and (c) graphs for the viruses H9N2-WB-1057183, H9N2-WB-1057209, and H9N2-JH-124248, respectively. The fluorescence values (in relative fluorescence units) plotted on the Y axis, concentration of oseltamivir carboxylate used in the assay plotted on the X axis. (d) and (e) Graphs of the reference strains A/Fukui/20/2004 (H3N2) and A/Fukui/45/2004 119 V Mutant (H3N2), susceptible and resistant controls, respectively.
H9N2 viruses, H9N2-WB-1057183 and H9N2-JH-124248, which had IC50 values of 0.72 nM and 0.26 nM, respectively. The in vitro and the allantoic methods showed significant virus inhibition. This could be because of the optimum interaction of
virus and drug when treated in vitro. This was also reflected in the virus growth inhibition when the virus and the drug were inoculated at the same time in the allantoic cavity of eggs. It was found that there was no need of increased time points for virus–drug
Table 2 Comparison of mean log HA titers of drug-treated and untreated H9N2 viruses (by in vitro treatment of virus and drug). Dilution of virusa
Mean log HA titers of AI H9N2 virus strains H9N2-WB-1057183 Untreatedb
Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 10-9 a
2.58 2.36 2.68 2.91 1.83 2.53 2.11 2.67 1.76 0.96
± ± ± ± ± ± ± ± ± ±
0.4 0.4 0.4 0.2 1.3 0.9 1.3 0.3 1.2 1.3
H9N2-WB-1057209 Treatedc 0.55 1.90 1.88 1.12 0.49 0.21 0.07 0.11 0.12 0.19
± ± ± ± ± ± ± ± ± ±
0.7 0.8 0.6 1.0 0.8 0.5 0.3 0.5 0.6 0.6
Untreatedb 2.58 2.68 2.90 2.93 3.06 3.13 3.00 3.13 3.15 2.95
± ± ± ± ± ± ± ± ± ±
0.7 0.6 0.2 0.7 0.1 0.2 0.7 0.1 0.2 1.3
H9N2-JH-124248 Treatedc 2.08 2.54 2.82 2.81 2.08 1.09 0.74 0.09 0 0
± ± ± ± ± ± ± ± ± ±
0.8 0.7 0.5 0.6 1.3 1.3 1.2 0.4 0 0
Untreatedb 2.54 2.34 2.72 2.67 2.60 2.49 2.70 2.55 2.49 1.55
± ± ± ± ± ± ± ± ± ±
Virus diluted in phosphate buffered saline. Mean log HA titers (± Standard deviation) of untreated controls, pooled data for three experiments. c Mean log HA titers of eggs treated with 14 g/ml of oseltamivir carboxylate (± Standard deviation), pooled data for three experiments. The treated groups showing significant difference as compared to the untreated ones (p < 0.05) depicted by boldface font. b
0.5 0.6 0.3 0.6 0.5 0.8 0.4 0.6 0.8 1.5
Treatedc 0.41 1.18 1.30 1.44 1.38 1.53 0.99 0.90 0.16 0.33
± ± ± ± ± ± ± ± ± ±
0.5 0.9 1.1 1.1 1.1 1.1 1.2 1.2 0.6 0.8
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interaction during such studies. Unlike the earlier study, the albumen route was not effective and did not show significant virus inhibition (Wang et al., 2008). This could be due to incomplete dissemination of OC when inoculated by the albumen route. However, dissemination of OC via the albumen route is not yet established, and studies on the absorption of OC in eggs would give more idea about its distribution, which needs further investigation. It has been shown that the albumen route should be preferred for drug delivery if the drug has low oral bioavailability (Wang et al., 2008). Since OC has a bioavailability of approximately 80% (Hayden, 2005), the albumen route was probably not suitable. The NAI assay is widely used for testing susceptibility of influenza viruses against the neuraminidase inhibitors zanamivir and oseltamivir. The assay is expensive and requires standard reference strains for avian influenza viruses, which are not available. In the present study, A/Fukui/20/2004 (wild type H3N2) and A/Fukui/45/2004 (variant type H3N2) were used as reference standards and they were found useful for testing the AI H9N2 virus. It has been reported that H274Y, R292K, E 119 V, and N294S mutations in the NA gene of influenza viruses confer resistance to oseltamivir (Collins et al., 2009). The sequences of the tested H9N2 virus did not show presence of these resistance markers (data not shown). The advantages of the use of embryonated chicken egg as a model over the NAI assay and prediction using the molecular signatures of virus are that the present study involves host and pathogen interaction. However, comprehensive studies involving NAI assay, molecular signatures of viruses, and using the egg model can provide a solid basis for predicting antiviral susceptibility or resistance. The limitation of the present study is that other AI viruses were not used. Therefore, there is a need to assess this model further for other high and low pathogenic AI viruses. Also, studies using prolonged incubation of inoculated eggs beyond 72 h need further evaluation. Thus, in view of emerging influenza viruses, the egg model using the “in vitro” and “allantoic route” methods could be explored for testing antiviral drugs against AI viruses. Declarations Role of the funding source: The funding agency does not have any role in study design, collection, analysis, and interpretation of data, in the writing of the report or in the decision to submit the article for publication. Conflict of Interest: We do not have any conflict of interests. Acknowledgments We thank Hoffmann-La Roche (Basel, Switzerland) for providing oseltamivir carboxylate, SS Kode for help in laboratory work, SM Jadhav, AM Walimbe for statistical analysis, CS Mote for histopathology studies, Aeron C Hurt, WHO Influenza Collaborating Centre, Melbourne, Australia, for reference strains used in the NAI assay. We thank DT Mourya, Director, National Institute of Virology (NIV), Pune, India and MS Chadha, for encouragement and support. This work was supported by the intramural funds, Indian Council of Medical Research, Department of Health Research, Ministry of Health and Family Welfare, Government of India. References Butt, K.M., Smith, G.J.D., Chen, H., Zhang, L.J., Leung, H.C., Xu, K.M., Lim, W., Webster, R.G., Yuen, K.Y., Peiris, J.S.M., Guan, Y., 2005. Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003. J. Clin. Microbiol. 43, 5760–5767.
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Use of embryonated chicken egg as a model to study the susceptibility of avian influenza H9N2 viruses to oseltamivir carboxylate Deeksha S. Tare, Shailesh D. Pawar ∗ National Institute of Virology-Microbial Containment Complex, 130/1, Sus Road, Pashan, Pune 411021, India
a b s t r a c t Article history: Received 11 January 2015 Received in revised form 13 August 2015 Accepted 13 August 2015 Available online 20 August 2015 Keywords: Antivirals Oseltamivir Avian influenza H9N2 Embryonated chicken eggs
Avian influenza (AI) H9N2 viruses are endemic in many bird species, and human infections of H9N2 viruses have been reported. Oseltamivir phosphate (Tamiflu® ) is the available antiviral drug for the treatment and prophylaxis of influenza. There are no reports of use of embryonated chicken egg as a model to study susceptibility of AI viruses to oseltamivir carboxylate (OC), the active metabolite. The present study was undertaken to explore the use of embryonated chicken eggs as a model for testing OC against the AI H9N2 viruses. A total of three AI H9N2 viruses, isolated in poultry in India, were used. Various virus dilutions were tested against 14 g/ml of OC. Three methods, namely (1) the in vitro virus–drug treatment, (2) drug delivery and virus challenge by allantoic route, and (3) drug delivery by albumen route and virus challenge by allantoic route were explored. The viruses were also tested using the fluorescence-based neuraminidase inhibitor (NAI) assay. There was significant inhibition (p < 0.05) of the H9N2 viruses in presence of OC. The infectious virus titers as well as hemagglutination titers were significantly lower in presence of OC as compared to controls. The in vitro treatment of virus and drug; and drug and virus delivery at the same time by allantoic route showed significantly higher inhibition (p < 0.05) of virus growth than that by the albumen route. In the NAI assay, the half maximal inhibitory concentration (IC50 ) values of the H9N2 viruses were within the standard range for known susceptible reference virus. In conclusion, the H9N2 viruses used in the study were susceptible to OC. Embryonated chicken egg could be used as a model to study susceptibility of AI viruses to antiviral drugs. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Influenza viruses are single stranded, negative-sense RNA viruses, which belong to the family Orthomyxoviridae. The world experienced the most recent influenza pandemic in the year 2009 caused by the H1N1 virus. Avian influenza (AI) viruses such as H5N1, H7N9, and H9N2 are emerging and are potential threats to human and animal health. In addition to this, there are reports of resistance of influenza viruses to the antiviral drugs such as oseltamivir (Monto et al., 2006). Therefore, it is necessary to screen existing as well as emerging influenza viruses against the available antiviral agents. Oseltamivir (oseltamivir phosphate) (Tamiflu® ) is an orally administered antiviral drug, recommended by the World Health Organization (WHO) for use in the clinical management of pandemic and seasonal influenza virus infections of varying severity.
∗ Corresponding author. E-mail address: [email protected] (S.D. Pawar). http://dx.doi.org/10.1016/j.jviromet.2015.08.009 0166-0934/© 2015 Elsevier B.V. All rights reserved.
Oseltamivir carboxylate (OC) is the active metabolite of oseltamivir, and is a transition-state analog of sialic acid that is a potent selective inhibitor of influenza A and B virus neuraminidases (Hayden, 2005). There have been increasing speculations about the pandemic potential of AI H9N2 viruses (Paul, 2008). There are reports of prevalence of H9N2 viruses in avian species from several parts of the world, and also from poultry in India (Pawar et al., 2012a; Nagarajan et al., 2009). Human infections of H9N2 virus have been reported from China, Hong Kong, and Egypt (Peiris et al., 1999; Butt et al., 2005; World Health Organization, 2015), and seroprevalence of antibodies against H9N2 viruses has also been reported (Xiong et al., 2014; Zhou et al., 2014; Huang et al., 2013; Pawar et al., 2012b; Hadipour, 2011; Guo et al., 1999). The existing methods for studying the susceptibility of influenza viruses to oseltamivir include use of neuraminidase inhibition assay and analysis of neuraminidase (NA) gene sequence for the markers of drug resistance to the drug (Hurt et al., 2004; McKimm-Breschkin, 2000). The use of tissue culture, mice, and ferrets for antiviral studies on influenza viruses has been reported
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(Govorkova et al., 2001; Govorkova et al., 2007). Moreover, no correlation between the susceptibility of H5N1 viruses by NAI assay and the protection offered by oseltamivir in mice (in vivo) was found (Govorkova et al., 2009). Embryonated chicken eggs have been conventionally used for isolation and propagation of influenza viruses (World Health Organization, 2002). The antiviral activities of amantidine, rimantidine, and zanamivir against influenza viruses have been studied using the egg model (Haertl et al., 2004; Sauerbrei et al., 2006). In the reported studies, the drug delivery was via the albumen route and virus challenge was by the chorioallantoic membrane. The use of the egg model by exploring the allantoic route for virus inoculation and the albumen route for drug delivery has been reported (Wang et al., 2008). However, there are no studies comparing various routes for drug delivery of OC in eggs for AI viruses. The present work was undertaken to study the susceptibility of three isolates of AI H9N2 viruses from India to OC, and to explore three methods for virus and drug treatment and delivery using embryonated chicken egg as a model. 2. Materials and methods 2.1. Viruses used AI H9N2 viruses isolated from India, namely, A/chicken/India/ WB-NIV1057183/2010 (H9N2) [H9N2-WB-1057183] (GenBank: JX310069), A/chicken/India/WB-NIV1057209/2010 (H9N2) [H9N2-WB-1057209], and A/chicken/India/JH-NIV 124248/2012 (H9N2) [H9N2-JH-124248], were used in the study. 2.2. Virus propagation and detection The virus stock was prepared in embryonated chicken eggs by inoculating the virus by the allantoic route. The eggs used in all the experiments were 10-days-old at the time of inoculation and 13-days-old at the time of completion of experiment. The eggs were incubated for 72 h at 37 ◦ C, and were observed daily. After completion of the incubation, the embryos were chilled overnight at 4 ◦ C. The allantoic fluid was harvested, and hemagglutination (HA) assay was performed using 0.5% turkey red blood cells (World Health Organization, 2002). The virus stock was stored at −80 ◦ C till further use. The clinical end point was detection of virus by HA assay as a measure of virus growth in eggs. The study was conducted in accordance with the institutional guidelines. 2.3. Preparation of antiviral drug stock solution and toxicity testing The antiviral drug OC was kindly provided by Hoffmann-La Roche, Basel, Switzerland. A suspension of the drug was made in phosphate buffered saline (PBS) (pH 7.2). Peak plasma concentration of OC after a 75 mg dose in humans has been reported to be 0.35 g/ml (Hayden, 2005). Toxicity of OC was tested in eggs by inoculating 14 g/ml, 28 g/ml, 56 g/ml, and 112 g/ml of OC. The inoculated eggs were observed for 72 h. The embryos were decapitated, and fixed in formalin for a minimum period of 48 hs for histopathological analysis. Whole sections of the embryos were processed for paraffin embedding, sectioned at 4 m and stained with hematoxylin and eosin; and were examined for histopathological changes. 2.4. 50% egg infectious dose in presence and absence of OC The 50% egg infectious dose (EID50 ) titer of H9N2 virus was determined in the presence and absence of 14 g/ml of OC. The
Fig. 1. Schematic diagram of 10-days-old embryonated chicken egg showing the allantoic and albumen routes used for virus and drug inoculation.
virus was serially diluted tenfold (undiluted to 10−9 ) and treated by the in vitro drug treatment method (Song et al., 2007; Rajik et al., 2009). Each dilution was inoculated in ten eggs. Eggs showing HA titer ≥ 2 HA units were considered positive, while those showing no titer were negative. The EID50 was calculated using the Reed and Muench method (Reed and Muench, 1938). The mean log HA titers for individual dilutions were also compared. The experiment was performed three times. 100 EID50 virus was then used for the further experiments using three different methods.
2.5. In ovo antiviral assays Various drug concentrations of OC, 1.75 g/ml, 3.5 g/ml, 7 g/ml, 14 g/ml, 28 g/ml, 56 g/ml, and 112 g/ml were tested against 100 EID50 virus, using the in vitro drug treatment method (described below), to determine the concentration of drug required for complete inhibition of virus growth. Each drug concentration was inoculated in ten eggs. Drug concentrations 1.75 g/ml, 3.5 g/ml, and 7 g/ml did not show any significant drop in the virus HA titers after treatment as compared to the untreated controls (p > 0.05). OC concentrations of 14 g/ml and above showed complete inhibition of the virus. Therefore, 14 g/ml of OC was used for the in ovo antiviral assays. The following three methods were then used for the in ovo antiviral assays: (a) In vitro treatment of virus and drug: Equal volumes of the drug and virus were mixed and incubated for 1 h at 37 ◦ C. This mixture (0.2 ml virus + 0.2 ml drug) was inoculated into the allantoic cavity. (b) Virus and drug delivery by the allantoic route: In the allantoic cavity, 0.2 ml virus suspension was inoculated. In addition, 0.2 ml drug was administered at two time points [namely 0 h (ALL 0 h) and 2 h (ALL 2 h)] by the allantoic route. (c) Drug delivery by the albumen route: In the allantoic cavity, 0.2 ml virus was inoculated and 0.2 ml drug was administered, through albumen at two time points; 0 h (ALB 0 h) and 2 h (ALB 2 h) (Fig. 1) (Wang et al., 2008). Each treatment group, including the virus control group, consisted of ten eggs. There was no drug administration in the virus controls, PBS was used instead of OC. Total controls, which had no virus or drug inoculated, were also included in each experiment. The inoculated eggs were incubated at 37 ◦ C for 72 h. The eggs were observed after an interval of 24 h till 72 h. At the end of 72 h, the eggs were chilled at 4 ◦ C overnight. The allantoic fluids were harvested, and tested by the HA assay.
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Table 1 50% egg infectious dose (EID50 ) titers of drug-treated and untreated H9N2 viruses. AI H9N2 virus strains
H9N2-WB-1057183
Egg infectious dose 50% (EID50 ) titersa Untreatedb
Treatedc
107.57
103.40 103.47
107.61 109.39 H9N2-WB-1057209
H9N2-JH-124248
1010.00 1010.00
103.30 104.35 106.36
109.45 108.42
104.31 103.70
109.84 109.49
104.54 106.10
a 50% infectious titers calculated by the Reed and Muench method. Virus dilutions from undiluted to 10−10 . Each experiment was performed three times. b Untreated controls. No oseltamivir carboxylate was used. c Treated with 14 g/ml oseltamivir carboxylate. The treated groups showing significant difference as compared to the untreated ones (p < 0.05) depicted by boldface font (Independent sample t-test)
2.6. Statistical analysis
Fig. 2. Inhibition of virus growth by oseltamivir carboxylate using three treatment methods. The mean of log HA titers of the allantoic fluids harvested from the treated and untreated groups plotted against the various treatment methods used. In vitro drug–virus treatment method, and inoculation of the drug and virus at the same time by allantoic route (ALL 0 h) showed significant inhibition (p < 0.05) in the virus titers.
Independent sample t-test for the comparison of virus growth inhibition between treated and untreated groups was performed in the software “SPSS” (IBM). The log mean HA titers from inoculated eggs were calculated in “Microsoft Excel” and the difference in the HA titers was determined by t-test in the “EpiInfo” software (Centers for Disease Control, USA).
virus by the in vitro treatment method. The other methods of treatment, namely the ALL 2 h, ALB 0 h, and ALB 2 h showed inhibition as compared to the untreated controls, but it was not significant when compared to the in vitro and ALL 0 h treatment methods (Fig. 2).
2.7. Fluorescent neuraminidase inhibition (NAI) assay The inhibitory effect of OC on the viruses was also tested by the fluorescence-based NAI assay as per the method described by Hurt et al, 2004 to screen susceptibility or resistance. The viruses A/Fukui/20/2004 wild type H3N2 and A/Fukui/45/2004 (H3N2) 119 V mutant (kindly provided by Dr. Aeron Hurt at the WHO Collaborating Center for Reference and Research on Influenza, Melbourne, Australia) were used as reference standards for susceptibility and resistance, respectively. The plots and the IC50 values were calculated for the samples and standards by using the curve fitting software JASPR (CDC, USA). 3. Results
3.4. Fluorescent NAI assay The IC50 values of all the three H9N2 viruses were 0.72 nM, 3.66 nM, and 0.26 nM for H9N2-WB-1057183, H9N2-WB-1057209, and H9N2-JH-124248 viruses, respectively, which were in the range (0.01 nM–5.0 nM) of the OC sensitive standard strain A/Fukui/20/2004 (H3N2). This indicated that all three tested H9N2 viruses were sensitive to OC. H9N2-WB-1057209 virus showed the higher IC50 value among the three viruses. The reference resistant strain A/Fukui/45/2004 (H3N2) had IC50 values in the range 23 nM–378 nM (Fig. 3) (International Society for Influenza and other Respiratory Virus Diseases (ISIRV) Antiviral group (AVG)).
3.1. Toxicity testing
4. Discussion
There was no toxicity of OC in the eggs, as indicated by the absence of mortality and abnormalities in the histopathology of the embryos.
Oseltamivir phosphate has been shown to be effective in the experimental infection of mice by H9N2 virus, and also by using a modified enzyme-linked immunoassay and NAI assays (Leneva et al., 2000; Govorkova et al., 2001). However, there are no studies exploring various routes of virus and drug delivery in eggs for testing OC against AI H9N2 viruses. The previous studies in eggs used human viruses, and the drug oseltamivir phosphate and not the active OC (Heartl et al., 2004; Sauerbrei et al., 2006; Wang et al., 2008). However, in the present study, OC, which is the active metabolite form, has been used. In ovo experiments are at the borderline between in vitro and in vivo studies, and hence do not conflict with ethical and legal aspects of animal protection (Sauerbrei et al., 2006; Wang et al., 2008) When infectious virus titers (EID50 ) of three viruses with and without OC were compared (Table 2), H9N2-WB-1057209 virus dilutions from 10−1 to 10−3 did not show significant inhibition in the mean log titers. This might be due to the fact that this virus showed higher IC50 value i.e., 3.66 nM, as compared to the other two
3.2. 50% egg infectious dose in presence and absence of OC When the various dilutions of the three viruses were subjected to treatment (by the in vitro drug virus treatment method), there was significant reduction (p < 0.05) in the infectious virus titers (EID50 ) and also in the mean log HA titers in the presence of OC, as compared to controls (Table 1, 2). 3.3. In ovo antiviral assays In the further experiments for exploring different routes, the in vitro drug–virus treatment method, and inoculation of the drug and virus at the same time by allantoic route (ALL 0hr) showed significant inhibition (p < 0.05) of the virus, as compared to the other drug treatment methods. There was a complete inhibition of the
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Fig. 3. Half maximal inhibitory concentration (IC50 ) of H9N2 viruses in neuraminidase inhibition assay. Output graphs obtained for the neuraminidase inhibition assay, from the software JASPR. (a), (b), and (c) graphs for the viruses H9N2-WB-1057183, H9N2-WB-1057209, and H9N2-JH-124248, respectively. The fluorescence values (in relative fluorescence units) plotted on the Y axis, concentration of oseltamivir carboxylate used in the assay plotted on the X axis. (d) and (e) Graphs of the reference strains A/Fukui/20/2004 (H3N2) and A/Fukui/45/2004 119 V Mutant (H3N2), susceptible and resistant controls, respectively.
H9N2 viruses, H9N2-WB-1057183 and H9N2-JH-124248, which had IC50 values of 0.72 nM and 0.26 nM, respectively. The in vitro and the allantoic methods showed significant virus inhibition. This could be because of the optimum interaction of
virus and drug when treated in vitro. This was also reflected in the virus growth inhibition when the virus and the drug were inoculated at the same time in the allantoic cavity of eggs. It was found that there was no need of increased time points for virus–drug
Table 2 Comparison of mean log HA titers of drug-treated and untreated H9N2 viruses (by in vitro treatment of virus and drug). Dilution of virusa
Mean log HA titers of AI H9N2 virus strains H9N2-WB-1057183 Untreatedb
Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 10-9 a
2.58 2.36 2.68 2.91 1.83 2.53 2.11 2.67 1.76 0.96
± ± ± ± ± ± ± ± ± ±
0.4 0.4 0.4 0.2 1.3 0.9 1.3 0.3 1.2 1.3
H9N2-WB-1057209 Treatedc 0.55 1.90 1.88 1.12 0.49 0.21 0.07 0.11 0.12 0.19
± ± ± ± ± ± ± ± ± ±
0.7 0.8 0.6 1.0 0.8 0.5 0.3 0.5 0.6 0.6
Untreatedb 2.58 2.68 2.90 2.93 3.06 3.13 3.00 3.13 3.15 2.95
± ± ± ± ± ± ± ± ± ±
0.7 0.6 0.2 0.7 0.1 0.2 0.7 0.1 0.2 1.3
H9N2-JH-124248 Treatedc 2.08 2.54 2.82 2.81 2.08 1.09 0.74 0.09 0 0
± ± ± ± ± ± ± ± ± ±
0.8 0.7 0.5 0.6 1.3 1.3 1.2 0.4 0 0
Untreatedb 2.54 2.34 2.72 2.67 2.60 2.49 2.70 2.55 2.49 1.55
± ± ± ± ± ± ± ± ± ±
Virus diluted in phosphate buffered saline. Mean log HA titers (± Standard deviation) of untreated controls, pooled data for three experiments. c Mean log HA titers of eggs treated with 14 g/ml of oseltamivir carboxylate (± Standard deviation), pooled data for three experiments. The treated groups showing significant difference as compared to the untreated ones (p < 0.05) depicted by boldface font. b
0.5 0.6 0.3 0.6 0.5 0.8 0.4 0.6 0.8 1.5
Treatedc 0.41 1.18 1.30 1.44 1.38 1.53 0.99 0.90 0.16 0.33
± ± ± ± ± ± ± ± ± ±
0.5 0.9 1.1 1.1 1.1 1.1 1.2 1.2 0.6 0.8
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interaction during such studies. Unlike the earlier study, the albumen route was not effective and did not show significant virus inhibition (Wang et al., 2008). This could be due to incomplete dissemination of OC when inoculated by the albumen route. However, dissemination of OC via the albumen route is not yet established, and studies on the absorption of OC in eggs would give more idea about its distribution, which needs further investigation. It has been shown that the albumen route should be preferred for drug delivery if the drug has low oral bioavailability (Wang et al., 2008). Since OC has a bioavailability of approximately 80% (Hayden, 2005), the albumen route was probably not suitable. The NAI assay is widely used for testing susceptibility of influenza viruses against the neuraminidase inhibitors zanamivir and oseltamivir. The assay is expensive and requires standard reference strains for avian influenza viruses, which are not available. In the present study, A/Fukui/20/2004 (wild type H3N2) and A/Fukui/45/2004 (variant type H3N2) were used as reference standards and they were found useful for testing the AI H9N2 virus. It has been reported that H274Y, R292K, E 119 V, and N294S mutations in the NA gene of influenza viruses confer resistance to oseltamivir (Collins et al., 2009). The sequences of the tested H9N2 virus did not show presence of these resistance markers (data not shown). The advantages of the use of embryonated chicken egg as a model over the NAI assay and prediction using the molecular signatures of virus are that the present study involves host and pathogen interaction. However, comprehensive studies involving NAI assay, molecular signatures of viruses, and using the egg model can provide a solid basis for predicting antiviral susceptibility or resistance. The limitation of the present study is that other AI viruses were not used. Therefore, there is a need to assess this model further for other high and low pathogenic AI viruses. Also, studies using prolonged incubation of inoculated eggs beyond 72 h need further evaluation. Thus, in view of emerging influenza viruses, the egg model using the “in vitro” and “allantoic route” methods could be explored for testing antiviral drugs against AI viruses. Declarations Role of the funding source: The funding agency does not have any role in study design, collection, analysis, and interpretation of data, in the writing of the report or in the decision to submit the article for publication. Conflict of Interest: We do not have any conflict of interests. Acknowledgments We thank Hoffmann-La Roche (Basel, Switzerland) for providing oseltamivir carboxylate, SS Kode for help in laboratory work, SM Jadhav, AM Walimbe for statistical analysis, CS Mote for histopathology studies, Aeron C Hurt, WHO Influenza Collaborating Centre, Melbourne, Australia, for reference strains used in the NAI assay. We thank DT Mourya, Director, National Institute of Virology (NIV), Pune, India and MS Chadha, for encouragement and support. This work was supported by the intramural funds, Indian Council of Medical Research, Department of Health Research, Ministry of Health and Family Welfare, Government of India. References Butt, K.M., Smith, G.J.D., Chen, H., Zhang, L.J., Leung, H.C., Xu, K.M., Lim, W., Webster, R.G., Yuen, K.Y., Peiris, J.S.M., Guan, Y., 2005. Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003. J. Clin. Microbiol. 43, 5760–5767.
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